The present study examined the long-standing concept that changes in hippocampal circuitry contribute to age-related learning impairment. Individual differences in spatial learning were documented in young and aged Long-Evans rats by using a hippocampal-dependent version of the Morris water maze. Postmortem analysis used a confocal laser-scanning microscopy method to quantify changes in immunofluorescence staining for the presynaptic vesicle glycoprotein, synaptophysin (SYN), in the principal relays of hippocampal circuitry. Comparisons based on chronological age alone failed to reveal a reliable difference in the intensity of SYN staining in any region that was examined. In contrast, aged subjects with spatial learning deficits displayed significant reductions in SYN immunoreactivity in CA3 lacunosum-moleculare (LM) relative to either young controls or age-matched rats with preserved learning. SYN intensity values for the latter groups were indistinguishable. In addition, individual differences in spatial learning capacity among the aged rats correlated with levels of SYN staining selectively in three regions: outer and middle portions of the dentate gyrus molecular layer and CA3-LM. The cross-sectional area of SYN labeling, by comparison, was not reliably affected in relation cognitive status. These findings are the first to demonstrate that a circuit-specific pattern of variability in the connectional organization of the hippocampus is coupled to individual differences in the cognitive outcome of normal aging. The regional specificity of these effects suggests that a decline in the fidelity of input to the hippocampus from the entorhinal cortex may play a critical role. Key words: circuit organization; synaptophysin; hippocampus; entorhinal cortex; aging; spatial learning; Morris water mazeA substantial proportion of aged individuals exhibit learning and memory deficits that qualitatively resemble the effects of direct hippocampal damage (for review, see Gallagher and Rapp, 1997). Related alterations are observed in hippocampal neuronal activity in aged rats with spatial learning deficits, including a decline in the scope of information controlling location-specific firing, and modified place field stability Shen et al., 1997; Tanila et al., 1997a,b). Against this background recent stereological investigations indicate that the total number of dentate gyrus granule cells and pyramidal neurons in fields CA3/2 and CA1 remains stable in aged mice (Calhoun et al

Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT2C receptor (5-HT2CR) is essential in normal physiology, whereas aberrant 5-HT2CR function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT2CR interacts with specific protein partners, but the impact of such interactions on 5-HT2CR function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT2CR and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT2CR-mediated biology but not that of the closely homologous 5-HT2AR. A peptide derived from the third intracellular loop of the human 5-HT2CR [3L4F (third loop, fourth fragment)] disrupted the association, allosterically augmented 5-HT2CR-mediated signaling in live cells, and acted as a positive allosteric modulator in rats in vivo. We identified the critical residues within an 8 aa fragment of the 3L4F peptide that maintained efficacy (within the picomolar range) in live cells similar to that of the 3L4F peptide. Last, molecular modeling identified key structural features and potential interaction sites of the active 3L4F peptides against PTEN. These compelling data demonstrate the specificity and importance of this protein assembly in cellular events and behaviors mediated by 5-HT2CR signaling and provide a chemical guidepost to the future development of drug-like peptide or small-molecule inhibitors as neuroprobes to study 5-HT2CR allostery and therapeutics for 5-HT2CR-mediated disorders.

Hippocampal N-methyl-D-Aspartate (NMDA) receptors mediate mechanisms of cellular plasticity critical for spatial learning in rats. The present study examined the relationship between spatial learning and NMDA receptor expression in discrete neuronal populations, as well as the degree to which putative age-related changes in NMDA receptors are coupled to the effects of normal aging on spatial learning. Young and aged Long-Evans rats were tested in a Morris water maze task that depends on the integrity of the hippocampus. Levels of NR1, the obligatory subunit for a functional NMDA receptor, were subsequently quantified both biochemically by Western blot in whole homogenized hippocampus, and immunocytochemically by using a high-resolution confocal laser scanning microscopy method. The latter approach allowed comprehensive, regional analysis of discrete elements of excitatory hippocampal circuitry. Neither method revealed global changes, nor were there region-specific differences in hippocampal NR1 levels between young and aged animals. However, across all subjects, individual differences in spatial learning ability correlated with NR1 immunofluorescence levels selectively in CA3 neurons of the hippocampus. Parallel confocal microscopic analysis of the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor failed to reveal reliable differences as a function of age or spatial learning ability. This analysis linking age, performance, and NR1 levels demonstrates that although dendritic NR1 is generally preserved in the aged rat hippocampus, levels of this receptor subunit in selective elements of hippocampal circuitry are linked to spatial learning. These findings suggest that NMDA receptor abundance in CA3 bears a critical relationship to learning mediated by the hippocampus throughout the life span.

It is now well accepted that at least some serotonin receptors exist in dimeric and oligmeric forms. The linking of receptor ligands has been shown to have potential in the development of selective agonists and antagonists for traditionally refractive receptors. Here we report the development of a dimeric version of the known 5-HT2AR antagonist, M-100907. Derivatives of M-100907 were synthesized to determine an appropriate site for the linker connection. Then, homodimers with polyether linkers of different lengths were functionally tested in a bioassay to determine the optimal linker length. Attachment at the catechol of M-100907 with linkers between 12 and 18 atoms in length proved to be optimal.

Allosteric modulators of the serotonin (5-HT) 5-HT 2C receptor (5-HT 2C R) present a unique drug design strategy to augment the response to endogenous 5-HT in a site-and event-specific manner with great potential as novel central nervous system probes and therapeutics. To date, PNU-69176E is the only reported selective positive allosteric modulator for the 5-HT 2C R. For the first time, an optimized synthetic route to readily access PNU-69176E (1) and its diastereomer 2 has been established in moderate to good overall yields over 10 steps starting from commercially available picolinic acid. This synthetic approach not only enables a feasible preparation of a sufficient amount of 1 for use as a reference compound for secondary pharmacological studies, but also provides an efficient synthesis of key intermediates to develop novel and simplified 5-HT 2C R allosteric modulators. Compound 1 and its diastereomer 2 were functionally characterized in Chinese hamster ovary (CHO) cells stably transfected with the 5-HT 2C R using an intracellular calcium (Ca i 2+ ) release assay. Compound 1 demonstrated efficacy and potency as an allosteric modulator for the 5-HT 2C R with no intrinsic agonist activity. Compound 1 did not alter 5-HT-evoked Ca i 2+ in CHO cells stably transfected with the highly homologous 5-HT 2A R. In contrast, the diastereomer 2 did not alter 5-HT-evoked Ca i 2+ release in 5-HT 2A R-CHO or 5-HT 2C R-CHO cells or exhibit intrinsic agonist activity.KEYWORDS: PNU-69176E, diastereomer, synthesis, allosteric modulator, 5-HT 2C receptor T he serotonin (5-HT) 2C receptor (5-HT 2C R) is implicated in a diversity of physiological functions, such as nociception, motor behavior, endocrine secretion, thermoregulation, appetite modulation, and the control of exchanges between the central nervous system (CNS) and the cerebrospinal fluid.1 This receptor has also been implicated in numerous psychiatric pathologies, and the modulation of 5-HT 2C R function holds a tremendous amount of therapeutic promise for the treatment of diseases of significant unmet medical need, including addiction, anxiety, depression, obesity/ eating disorders, Parkinson's disease, and schizophrenia.Successful development of 5-HT 2C R ligands requires selectivity versus the highly homologous 5-HT 2A R and 5-HT 2B R, as 5-HT 2A/2B R agonists can result in significant CNS (5-HT 2A R) and cardiovascular (5-HT 2B R) adverse effects.3 Allosteric modulators of 5-HT 2C R present a novel and attractive drug design strategy to augment the response to endogenous 5-HT and to achieve high receptor subtype selectivity and specificity with ligand binding to an allosteric site rather than to the orthosteric binding site that binds the endogenous agonist.